Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1

Abstract

Background In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. Methods We conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results. Results We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G). Conclusion We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.

The Site and Type of CLCN5 Genetic Variation Impact the Resulting Dent Disease-1 Phenotype

“…Variants within CLCN5 were classified according to the ACMG and American College of Pathologists guidelines. 20 All variants were described based on the transcript NM_001127898.4 (mRNA, 9863 base pairs, 15 exons [13 coding], and a 2451 base pairs coding sequence encoding an 816 aa protein), 21 as listed in gnomAD version 2.1.1. Exons are numbered from the first coding exon.…”

Genetics and phenotypic heterogeneity of Dent disease: the dark side of the moon

“…Several other researchers investigated how CLCN5 mutations reflect on ClC-5 activity and/or plasma membrane expression (Yamamoto et al 2000;Mo et al 2004;Grand et al 2011;Gorvin et al 2013;D'Antonio et al 2013;Satoh et al 2016;Tang et al 2016;Bignon et al 2018;Chang et al 2020), while others examined the changes due to mutations affecting CLCN5 splice sites (Forino et al 2004;Ramos-Trujillo et al 2007;Inoue et al 2020).…”

The Contribution of COL4A5 Splicing Variants to the Pathogenesis of X-Linked Alport Syndrome

“…However, the minigene assay has high flexibility because it does not require any mRNA or even gDNA samples from patients. Variants can be introduced to the minigene by using site directed mutagenesis allowing transcript analysis even if patient sample is not available (33,34). We have analyzed several novel intronic variants in various inherited kidney diseases using this assay (35)(36)(37)(38)(39)(40).…”